PROJECT SUMMARY Globally annual influenza epidemics are estimated to result in about 3 to 5 million cases of severe illness. Between 291,000 and 646,000 people worldwide die from seasonal influenza-related respiratory illnesses each year by the most recent report. The advances in sequencing technologies have led to the discovery of numerous long non-coding RNAs (lncRNAs). While the specific functions of these lncRNAs are still largely unknown, this new discovery offers an opportunity to develop novel classes of influenza interventions that target relevant lncRNAs or their interactions with other molecules. Our meta-analysis identified several lncRNAs that are predicted to be highly relevant to influenza A virus infection and host responses. Here we will extend these analyses with systematic investigations to establish their functional roles in influenza infection experimentally and to discover candidate lncRNAs as targets for influenza intervention. This project includes two Specific Aims: (1) experimentally establish how these highly ranked cellular lncRNAs affect influenza infection, and (2) identify lncRNA regulatory networks involved in influenza infection. In Aim 1, we will determine how influenza replication is altered when the expression of individual lncRNAs is knocked down or activated in human epithelial cells. For selected lncRNAs that the perturbation of their expressions affects influenza replication most effectively, we will confirm their effects on influenza infection in primary epithelial cells. In Aim 2, we will investigate: a) whether these highly ranked lncRNAs are interferon-stimulated genes; b) whether they act in cis or trans; c) the impact of selected lncRNAs on host responses to influenza infection. In particular, we will conduct an unbiased dual gene activation and knockout library screen to reconstruct the underlying lncRNA regulatory networks and uncover directional dependencies in these networks. Together, these analyses will allow us to better understand the functions of lncRNAs and their role in influenza infection, and to identify specific lncRNAs as novel targets for influenza intervention.